The traffic and fate of internalized G protein coupled receptors (GPCR) play a central role in the regulation of their function. Although the general pathways of GPCR internalization are shared among most members of the family, the precise mechanisms by which these pathways are regulated remain elusive. An extreme example of this is the Type I parathyroid hormone receptor (PTH1R). The PTH1R exhibits remarkable celland tissue-specific differences regarding the regulation of its activity and traffic by different ligands. Previous work from our laboratory and others has shown that the PTH1R is internalized by a combination of arrestindependent and independent processes. However, the relative contributions of these processes and the mechanisms that regulate them are still largely unknown. This application will test the hypothesis that PTH1R internalization is tightly regulated by the interactions of the PTH1R with PDZ (PSD95, discs-large, Zo-1) proteins. Abundant preliminary work presented here shows that the traffic the PTH1R is regulated by the PDZ proteins NHERF1 and Dvl2. This proposal will address the mechanisms by which these regulatory proteins perform their function in bone cell models. These studies will be conducted using state-of-the-art imaging (time-lapsed confocal and total internal reflection microscopy, single molecule tracking, and image correlation spectroscopy) in combination with classical membrane biochemistry and molecular biology strategies (analysis of the behavior of point mutants, ligand binding studies, immunoprecipitation techniques). A major innovation of the approach proposed is that it relies substantially on the analysis of individual endocytic steps to complement the global biochemical analyses. The tools developed and the conclusions obtained from this work will have applications to other members of the GPCR family.